Scholarly article on topic 'Vascular biology of ageing—Implications in hypertension'

Vascular biology of ageing—Implications in hypertension Academic research paper on "Clinical medicine"

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{"Vascular remodeling" / "Endothelial dysfunction" / "Oxidative stress" / Mitochondria}

Abstract of research paper on Clinical medicine, author of scientific article — Adam Harvey, Augusto C. Montezano, Rhian M. Touyz

Abstract Ageing is associated with functional, structural and mechanical changes in arteries that closely resemble the vascular alterations in hypertension. Characteristic features of large and small arteries that occur with ageing and during the development of hypertension include endothelial dysfunction, vascular remodelling, inflammation, calcification and increased stiffness. Arterial changes in young hypertensive patients mimic those in old normotensive individuals. Hypertension accelerates and augments age-related vascular remodelling and dysfunction, and ageing may impact on the severity of vascular damage in hypertension, indicating close interactions between biological ageing and blood pressure elevation. Molecular and cellular mechanisms underlying vascular alterations in ageing and hypertension are common and include aberrant signal transduction, oxidative stress and activation of pro-inflammatory and pro-fibrotic transcription factors. Strategies to suppress age-associated vascular changes could ameliorate vascular damage associated with hypertension. An overview on the vascular biology of ageing and hypertension is presented and novel molecular mechanisms contributing to these processes are discussed. The complex interaction between biological ageing and blood pressure elevation on the vasculature is highlighted. This article is part of a Special Issue entitled: CV Ageing.

Academic research paper on topic "Vascular biology of ageing—Implications in hypertension"

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YJMCC-08067; No. of pages: 10; 4C: 2,4, 6

Journal of Molecular and Cellular Cardiology xxx (2015) xxx-xxx

Contents lists available at ScienceDirect

Journal of Molecular and Cellular Cardiology

journal homepage: www.elsevier.com/locate/yjmcc

Review article

Vascular biology of ageing—Implications in hypertension

Adam Harvey, Augusto C. Montezano, Rhian M. Touyz *

Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, UK

ARTICLE INFO ABSTRACT

JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY

Article history:

Received 20 January 2015

Received in revised form 30 March 2015

Accepted 9 April 2015

Available online xxxx

Keywords:

Vascular remodeling Endothelial dysfunction Oxidative stress Mitochondria

Ageing is associated with functional, structural and mechanical changes in arteries that closely resemble the vascular alterations in hypertension. Characteristic features of large and small arteries that occur with ageing and during the development of hypertension include endothelial dysfunction, vascular remodelling, inflammation, calcification and increased stiffness. Arterial changes in young hypertensive patients mimic those in old normo-tensive individuals. Hypertension accelerates and augments age-related vascular remodelling and dysfunction, and ageing may impact on the severity of vascular damage in hypertension, indicating close interactions between biological ageing and blood pressure elevation. Molecular and cellular mechanisms underlying vascular alterations in ageing and hypertension are common and include aberrant signal transduction, oxidative stress and activation of pro-inflammatory and pro-fibrotic transcription factors. Strategies to suppress age-associated vascular changes could ameliorate vascular damage associated with hypertension. An overview on the vascular biology of ageing and hypertension is presented and novel molecular mechanisms contributing to these processes are discussed. The complex interaction between biological ageing and blood pressure elevation on the vasculature is highlighted. This article is part of a Special Issue entitled: CV Ageing.

© 2015 Published by Elsevier Ltd.

Contents

1. Introduction..............................................................................................................................0

2. Structural and mechanical changes in the ageing vasculature..................................................................................0

3. Vascular calcification......................................................................................................................0

4. Ageing associated vascular inflammation....................................................................................................0

5. Vascular contractility and ageing............................................................................................................0

6. Endothelial function and ageing............................................................................................................0

7. Vascular signalling in ageing..............................................................................................................0

7.1. Sirtuins............................................................................................................................0

7.2. PGC-1a............................................................................................................................0

7.3. FoxO transcription factors..........................................................................................................0

7.4. p66shc............................................................................................................................0

7.5. Cell cycle regulators, senescence and autophagy......................................................................................0

7.6. Mitogen-activated protein kinases (MAPK)............................................................................................0

7.7. Oxidative stress in vascular ageing..................................................................................................0

7.8. Endoplasmic reticulum stress in vascular ageing......................................................................................0

7.9. Vascular changes in hypertension recapitulate those in ageing..........................................................................0

7.10. Effects of pro-hypertensive stimuli on vascular ageing: the renin-angiotensin-aldosterone system (RAAS)............................0

7.11. Effects of novel anti-hypertensive factors on vascular ageing..........................................................................0

7.12. Vascular damage in hypertension may be independent of ageing......................................................................0

7.13. Lessons learned from children with hypertension....................................................................................0

8. Summary and conclusions................................................................................................................0

* Corresponding author at: Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow, G12 8TA, UK. Tel.: +44 141 330 7775/7774; fax: + 44141 330 3360. E-mail address: Rhian.Touyz@glasgow.ac.uk (R.M. Touyz).

http://dx.doi.org/10.1016/j.yjmcc.2015.04.011 0022-2828/© 2015 Published by Elsevier Ltd.

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Disclosures....................................................................................................................................0

Acknowledgements............................................................................................................................0

References....................................................................................................................................0

1. Introduction

Clinical studies show a significant relationship between ageing and increased blood pressure, with advancing age being a major non-modifiable risk factor in the development of hypertension [1]. This is due, in part, to changes that occur in the vasculature, including endothe-lial dysfunction, vascular remodelling, increased vascular stiffness and inflammation. These functional and structural changes define the 'vascular phenotype' of hypertension, features that are also found during ageing [2] (Fig. 1). At the cellular level, there is endothelial cell damage, increased vascular smooth muscle cell growth, cell migration, inflammation, contraction, extracellular matrix deposition, fibrosis, and calcification [3].

Young patients with elevated blood pressure exhibit arterial changes similar to those in older individuals with normal blood pressure, and accordingly the concept of 'premature' or 'early' vascular ageing in hypertension has been proposed [4]. Hypertension accelerates age-related vascular changes, processes that are attenuated when blood pressure is normalised. The direct relationship between ageing and vascular health is evident in progeria syndrome, where patients exhibit accelerated ageing, endothelial dysfunction, accelerated atherosclerosis and die prematurely from complications of cardiovascular disease, such as stroke and myocardial infarction [5]. Considering the fact that the population is ageing and that the major chronic disease of ageing is hypertension and associated cardiovascular complications, the potential health and economic burden in our modern society is enormous. Accordingly it is important to understand how vascular function changes with ageing and how this impacts on hypertension, so that targeted strategies could be developed to prevent and repair damaged 'aged' arteries and thereby reduce the risk of hypertension and target organ

Young - Healthy

f \ Normal Vascular Homeostasis

damage. In the present review, we discuss the vascular changes that occur with ageing and during the development of hypertension and focus on some molecular mechanisms that underlie these vascular changes.

2. Structural and mechanical changes in the ageing vasculature

Physiological changes to the vascular wall are dynamic and occur throughout life [6,7]. Endothelial cell turnover occurs over years, whereas that of vascular smooth muscle cells seems to occur over a shorter time period. Many structural and mechanical alterations have been observed in the aged vasculature including increased intimal-to-media (IM) thickness, evidenced by the finding that the IM thickness of the carotid artery increases two- to three-fold between 20 and 90 years of age [8,9]. Subclinical IM thickening is strongly associated with ageing and is also a predictor of future cardiovascular events [8,9]. Both aortic length and circumference gradually increase with advancing age [10-12]. Associated with these structural alterations are mechanical changes, characterised by a reduction in compliance, reduced elasticity/ distensibility and increased stiffness [8,9]. Stiffening of the large conduit arteries due to fracture of elastin fibres within the tunica media and collagenous remodelling, results in increased aortic pulse pressure and pulse wave velocity (PWV). Increased PWV, a non-invasive measure of vascular stiffness, increases in both sexes with ageing and is determined by the mean arterial pressure and the intrinsic stress/strain relationship (stiffness) of the arterial wall. As arterial wall stiffness increases, central systolic pressure increases and diastolic pressure decreases, leading to increased pulse pressure, an independent risk factor for future cardiovascular events [13]. Processes underlying these structural and mechanical changes involve growth and migration of vascular smooth muscle cells

Aged - Hypertension

r Endothelial dysfunction^ ^ M:L ratio Vascular remodelling Increased stiffness Vascular inflammation _Calcification J

Fig. 1. Schematic demonstrating vascular changes that occur during ageing and with the development of hypertension. Vascular changes in hypertension mimic those found in arteries observed with ageing.

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within the media, vascular calcification and changes in the ratio of collagen and elastin in the vascular wall. Physiologically, the ratio of collagen and elastin remains constant due to their gradual production and degradation. In aged rodents the absolute elastin content of the aorta was shown not to differ to young counterparts. However, increased collagen content in 30-month-old animals compared to 6-month-old animals meant that the relative elastin content was decreased [14]. Collagen and elastin are regulated by catabolic matrix metalloproteinases (MMPs). Throughout ageing the balance between MMPs and their inhibitors (TIMPs) changes. For example, increased MMP-2 expression and activity in the vessels of old rats and non-human primates is increased compared to young counterparts [15,16].

3. Vascular calcification

Vascular calcification is a tightly controlled process similar to bone formation, where mineralization of the internal elastic lamina and elastic fibres in the media results in vascular stiffening. Calcification of the vascular media is a hallmark of vascular ageing [17]. Upregulation of transcription factors such as cbfa1 (core-binding factor 1a)/Runx2, MSX-2 and bone morphogenetic protein 2 (BMP-2), are involved in normal bone development and vascular calcification by regulating the expression of osteogenic proteins, including osteocalcin, osteonectin, alkaline phosphatase, collagen-1, and bone sialoprotein [18,19]. Another mechanism contributing to vascular mineralization is loss of calcification inhibitors, such as fetuin-A, matrix Gla protein, pyrophosphate, and osteopontin [19-21]. Molecular processes underlying this remain to be fully defined but increased expression of BMP2 and the osteoblast transcription factor Runx2/Cbfa1 [22], and modulation of Ca2+ and Mg2+ transport through cation channels, such as TRPM7 may represent important mediators in this process [23,24]. A correlation between age and vascular calcification has been described from 5% in individuals younger than 50 years to > 12% in individuals older than 80 years [25]. Ageing-associated vascular calcification has been reported in the aorta of rodents where the associated mechanisms include dysregulation of Matrix Gla protein [26]. Further possible mechanisms contributing to increased calcification with ageing includes dysregulation of vascular pyrophosphate [27,28]. Human studies have shown a weak inverse correlation between age and plasma pyrophosphate [29,30].

4. Ageing associated vascular inflammation

With ageing there is a shift towards a proinflammatory vascular phenotype with upregulation of inflammatory cytokines, chemokines and adhesion molecules in the vascular wall [8,9,31-38]. Pro-inflammatory transcription factors and proteins that have been identified in the ageing vascular media include MCP-1, TGF-(31, MMP-2, AP-1 and NF-kB [8,9,39]. Expression and activation of these molecules increases with ageing, processes that are usually associated with increased generation of reactive oxygen species (ROS). In aged arteries, there is downregulation of the transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which stimulates expression of antioxidant enzymes, thereby leading to decreased anti-oxidant potential and increased ROS bioavailability with consequent oxidative stress [40]. Oxidative stress is a potent inducer of redox-sensitive pro-inflammatory signalling pathways, further contributing to inflammation and vascular damage with ageing [36-40].

5. Vascular contractility and ageing

Functionally, vascular contraction is altered during ageing and is determined in large part by changes in vascular smooth muscle cell cytoskeletal organisation and impaired contractile signalling. Mesenteric arteries from aged rats demonstrate hypercontractility in response to phenylephrine compared to young controls [40] an effect which is mirrored in the aorta [41]. These findings are paralleled in studies utilising

the senescence-accelerated mouse (SAM-P8), which demonstrate increased vascular contractility in response to phenylephrine [42]. Conversely, studies performed on carotid vessels from aged guinea pigs displayed reduced contractile response to both phenylephrine and endothelin-1 (ET-1) compared to younger controls [43]. Thus it appears that differential responses during ageing may differ between species.

At the cellular level, with ageing, vascular smooth muscle cells, which are normally contractile, undergo phenotypic changes to become stiff and pro-migratory. Subsets of apoptotic, senescent and proliferative cells as well as hyper-contractile cells may co-exist in the vascular media. A major trigger for these functional changes is an increase in intracellular free Ca2+ concentration ([Ca2+]i), which occurs following activation of phopholipase C (PLC) leading to the generation of second messengers insitol trisphosphate (IP3) and diacylglycerol (DAG) [39,44]. Ca2+ binds to calmodulin facilitating an interaction with myosin light chain kinase (MLCK) leading to its activation. Activated MLCK triggers phosphorylation of the regulatory light chains of myosin (MLC20) promoting cycling of myosin cross-bridges with actin and consequent contraction. Dephosphorylation of MLC20 by myosin light chain phosphatase (MLCP) results in VSMC relaxation. As such, the relative activities of MLCK and MLCP determine vascular smooth muscle tone by influencing the degree of MLC20 phosphorylation. Arteries from aged animals display altered responses to various contractile agents including norepinephrine, serotonin and KCL [44,45]. Mechanisms for this are incompletely understood, but the percentage of phosphorylated MLC20 induced by vasoactive agonists is different in young versus aged rats and may play a role in altered age-related contractile responses [45].

6. Endothelial function and ageing

The vascular endothelium is a monolayer of cells that lines blood vessels and plays a key role in arterial function through synthesis and release of biologically active molecules that can influence endothelial function in an autocrine or paracrine fashion. The healthy endothelium is characterised by a vasodilatory, anti-inflammatory and antithrombotic phenotype. Endothelial dysfunction is characterised by reduced vasodilatory responses to flow or agonists and is proinflammatory. Independent of the occurrence of other pathologies, ageing results in altered endothelium-dependent relaxation of both the aorta and resistance arteries in rodents [46,47]. These findings have been corroborated in human studies that suggest that endothelial function is gradually compromised with ageing [48,49]. A primary mechanism responsible for the deterioration of endothelial function with ageing is thought to be reduced bioavailability of the endothelium derived relaxing factor, nitric oxide (NO), due to its interaction with ROS to form peroxynitrite. Peroxynitrite oxidises BH4, an essential cofactor for NO synthesis by endothelial nitric oxide synthase (eNOS), to its inactive form resulting in reduced NO production. Furthermore, reduced BH4 can result in eNOS uncoupling whereby superoxide is produced in preference to NO. Reductions in BH4 levels have been reported in aged rodents [50].

7. Vascular signalling in ageing

Molecular mechanisms and cell signalling events underlying the structural and functional alterations observed during ageing are similar to those that occur in hypertension (Fig. 2). Many age/longevity-related molecules and signalling cascades have been described, of which a few of the novel systems are highlighted below.

7.1. Sirtuins

Sirtuins (SIRTs) are a family of NAD-dependent protein deacetylases and ribosyl transferases consisting of 7 members which are localised in the cytoplasm (SIRT1 and SIRT2), nucleus (SIRT1, SIRT2, SIRT 6 and SIRT 7) or mitochondria (SIRT3, SIRT4 and SIRT 5). SIRTs have been

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Pro-hypertensive Factors (RAS, ET-1, Aldosterone) Ï

i SIRT

î FOXO

Cell cycle regulators

MMP TIMP

Ca2+ Mg2+

Mitochondria dysfunction Noxactivation

p66Shc

îROS iNO

Inflammatory Genes MAPK

N / /\ I \ / \ I

Senescence Apoptosis Autophagy

Anoikis Proliferation

Mineralization

iVasorelaxation î Vasoreactivity

Vascularemodeling

Vascular calcification

Endothelial dysfunction

VCAM-1, ICAM-1 Cytokines Chemokines

Vascular inflammation

Hypertension

Fig. 2. Molecular and cellular mechanisms associated with vascular changes in ageing and hypertension. Activation of pro-fibrotic, pro-inflammatory, redox-sensitive and growth/apoptotic signalling pathways lead to changes in vascular structure, mechanics and function with resultant arterial remodelling, calcification, inflammation, stiffness and impaired vasoreactivity. These vascular alterations are common features during ageing and in hypertension. VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; MMP, matrix metallo-proteinases; TIMP, tissue inhibitor of metalloproteinase; RAS, renin angiotensin system; ET-1, endothelin-1 ; NO, nitric oxide.

238 implicated in various cellular processes associated with ageing, includ-

239 ing, apoptosis, inflammation and mitochondrial biogenesis. SIRTs are

240 able to modulate the ageing process in a number of species [51-53].

241 This is highlighted by the following: 1) SIRT1 protects against

242 phosphate-induced arterial calcification, possibly due to the inhibition

243 of osteoblastic transdifferentiation [54]; 2) mitochondrial localised

244 SIRT3 regulates many proteins that are important in the regulation of

245 mitochondrial function including pyruvate dehydrogenase, SOD2 and

246 cyclophilin D; 3) SIRT3 —/— mice exhibit accelerated cardiovascular

247 ageing [55] and 4) SIRT3 has vasoprotective effects through interaction

248 with FOXO3, which enhances mitochondrial antioxidant defence

249 systems [56].

250 7.2. PGC-la

251 Another emerging candidate implicated in age-related signalling in

252 the vasculature, is peroxisome proliferator-activated receptor gamma

253 coactivator-1a (PGC-1a), which plays an important role in regulating

254 mitochondrial biogenesis and turnover [57]. Because mitochondria re-

255 quire continuous recycling and regeneration throughout the lifespan

256 and are subject to continuous damage over time, regulation of mito-

257 chondrial biogenesis and turnover is critical for maintained energy

258 production and prevention of oxidative damage, and the promotion of

259 healthy ageing. Impaired mitochondrial biogenesis is an important in-

260 ducer of age-related changes in the endothelium and vascular smooth

261 muscle [58-60]. The aged vasculature displays reduced levels of PGC-

262 1a with consequent mitochondrial dysregulation of the electron trans-

263 port chain and other mitochondrial proteins leading to oxidative stress

264 and vascular injury [60]. Decreased AMPK activity may contribute to

reduced PGC-1a activation and impaired mitochondrial function associ- 265 ated with ageing [61]. 266

7.3. FoxO transcription factors 267

The FoxO family of Forkhead transcription factors are involved in 268 tumour suppression, energy metabolism, and longevity. Mammals ex- 269 press four FoxO isoforms, FoxO1, FoxO3, FoxO4 and FoxO6. FoxO1, 270 FoxO3 and FoxO4 are phosphorylated in an Akt-dependent manner 271 that promotes FoxO export from the nucleus to the cytoplasm, thereby 272 repressing FoxO transcriptional function. FoxO targets include genes 273 that have pivotal roles in cell cycle progression (p21, p27) and ROS de- 274 toxification (MnSOD) and thus may be important in regulation of the 275 ageing phenotype in the vasculature [62,63]. FoxO3 is a direct target 276 of SIRT3 deacetylation protecting mitochondria against age-related ox- 277 idative stress and promoting upregulation of genes that are essential 278 for mitochondrial homeostasis [64]. Several reports have suggested 279 that FoxO3 may be a determinant of ageing, due to the fact that 280 single-nucleotide polymorphisms in the FoxO3 gene are associated 281 with longevity in humans [65,66]. FoxO3 knockout mice however do 282 not exhibit reduced lifespan [67], and as such, the exact role of FoxO3 283 in longevity and ageing still remains unclear. 284

7.4. p66shc 285

Mitochondrial dysfunction and increased mitochondrial-derived 286 ROS have been implicated in vascular changes in ageing [68]. An impor- 287 tant mediator of mitochondrial ROS production and thus regulator of 288 the intracellular pathways that govern oxidative stress, apoptosis, and 289

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290 cell growth/survival is the adapter protein p66shc. p66Shc is phosphor-

291 ylated at serine 36 by PKQ3 and VEGF, resulting in recognition by the

292 prolyl isomerase Pin1, allowing translocation and entrance into mito-

293 chondria where it interacts with cytochrome C resulting in production

294 of H2O2. Levels of p66shc in heart, kidney and vascular smooth muscle

295 increase with ageing [69]. Mice lacking p66shc gene display a 30%

296 increase in lifespan compared to wild-type controls due to prevention

297 of oxidative stress and improved endothelial function [70,71 ].

298 7.5. Cell cycle regulators, senescence and autophagy

299 In culture, vascular cells respond to prolonged series of replication

300 and stresses by eventually entering an irreversible growth arrest or

301 senescent state [72]. After the Hayflick limit, cells enter an irreversible

302 cell cycle arrest in the G1 phase of the cell cycle and no longer respond

303 to growth stimuli. This phenomenon is called replicative senescence

304 and occurs in vascular ageing [73]. Senescent cells have a distinct

305 phenotype—they are large and flattened, express specific markers

306 ((-galactosidase), overexpress cell cycle molecular markers (p16 and

307 p21), form heterochromatic foci (yH2AX) and accumulate lipofuscin, a

308 non-degradable fluorescent compound [74]. Whilst the molecular mech-

309 anisms underlying cellular senescence have been the focus of numerous

310 studies, the impact of senescence in vivo has yet to be fully established,

311 especially since some studies show increased rates of vascular cell prolif-

312 eration in ageing and longevity [75,76].

313 Considering the remarkable plasticity of vascular smooth muscle

314 cells, there is a requirement for tight control of transcriptional, metabol-

315 ic and ultrastructural processes, events that are coordinated through

316 autophagy. Autophagy is the basic cellular mechanism that involves

317 cell degradation of unnecessary or dysfunctional molecules through

318 lysosomes [77]. In the vasculature, changes in autophagy have been

319 observed in experimental ageing [78].

320 7.6. Mitogen-activated protein kinases (MAPK)

321 Protein kinases are major regulators of signal transduction that

322 catalyse the phosphorylation of other proteins, thus regulating their ac-

323 tivity. Primary targets of protein kinases include transcription factors

324 which modulate intracellular signalling via specific alteration of down-

325 stream gene expression/activity [79]. A key group of protein kinases in

326 the vasculature are the serine/threonine sub-family, which act by pro-

327 moting phosphorylation of the OH group of serine or threonine residues

328 on target proteins [80]. Mitogen activated protein kinases (MAPKs)

329 represent a large family of proteins important in signal transduction

330 within the cardiovascular system, where they are involved in regulation

331 of a number of biological processes, such as cell migration, survival, ap-

332 optosis, proliferation, contraction and differentiation. MAPK signalling is

333 promoted by many stimuli including GPCR activation, receptor tyrosine

334 kinases, oxidative stress and growth factors, and comprises a number of

335 sequentially acting kinases which ultimately result in phosphorylation

336 and activation of terminal effector kinases, thereby transducing specific

337 cellular actions [80,81 ]. Several MAPK family subgroups have been iden-

338 tified, of which the major mammalian types appear to be ERK1/2, c-Jun

339 NH2-terminal kinases (JNK1, 2 and 3) and p38MAPK (a, (3, 8 and y),

340 which play key roles during cardiovascular development and vascular

341 function [82,83]. Several studies have demonstrated an age-dependent

342 increase in MAPK activation in vascular tissue [84,85].

343 77. Oxidative stress in vascular ageing

344 Common to many of the molecular and cellular processes described

345 above that underlie changes in the vasculature with ageing is oxidative

346 stress [86]. The concept that ROS are linked to ageing was suggested in

347 1 956 by Harman when he proposed the Free Radical Theory of Ageing,

348 stating that the accumulation of free radicals during ageing causes the

349 damage of biomolecules by these ROS and the development of

pathological disorders promoting cell senescence and organism ageing 350

[87,88]. Such processes are evident in vessels associated with ageing 351

and with hypertension [87,88]. Excessive production of ROS and reac- 352

tive nitrogen species (RNS) leads to oxidative modification of proteins, 353

DNA and lipids, which accumulate in cells leading to impaired cellular 354

and vascular function. In addition increased vascular ROS levels, togeth- 355 er with decreased eNOS-generated NO, compromise the vasodilatory 356

actions of NO and promote the formation of injurious peroxynitrite, 357

processes observed in aorta of aged rodents [89]. Oxidative stress is 358

critically involved in many of the molecular events of vascular ageing, 359

including: (1) increased pro-inflammatory responses in vascular cells, 360

(2) vascular dysfunction through oxidative modification of structural 361

and functional proteins regulating vascular contraction/relaxation, 362

fibrosis and calcification, (3) altered calcium homeostasis in vascular 363

cells, 4) activation of redox-sensitive pro-inflammatory and pro- 364

fibrotic transcription factors, and (4) activation of molecular mecha- 365 nisms leading to senescence and autophagy in endothelial and vascular 366

smooth muscle cells (Fig. 3). The fact that SOD mimetics, such as tempol, 367

normalise endothelial dysfunction in old rodents supports a role for in- 368 creased superoxide anion levels in age-related endothelial impairment 369

[90]. 370

Changes in cellular anti-oxidant systems are also important. The ex- 371

pression and activity of antioxidant enzymes, including SOD, decline as 372 tissues age. Decreased anti-oxidant capacity is further promoted by 373

downregulation of Nrf2, the master transcription factor regulating 374 anti-oxidant genes [91]. These processes are accompanied by chronic 375

low-grade inflammation mediated by redox-sensitive NFkB, which is 376

upregulated in aged vessels [92]. 377

Multiple oxidases generate ROS in the vascular wall and endotheli- 378

um, including NADPH oxidases (Nox), xanthine oxidase, uncoupled 379 NOS and mitochondrial oxidases. Of these, mitochondria seem to play 380

a major role in processes related to ageing. Noxs, of which there are 7 381

isoforms (Nox1-5, Duox1, Duox2), have also been shown to contribute 382

to oxidative stress in vascular ageing [93-95]. In particular, in aged 383

spontaneously hypertensive rat aortas, expression of Nox1 and Nox2, 384

but not of Nox4, was increased. This Nox upregulation was associated 385 with endothelial dysfunction, which was reversed by VAS2870, a Nox 386

inhibitor [96]. Noxs appear to be more important in pathological vascu- 387

lar remodelling associated with hypertension and cardiovascular 388

diseases [97-99]. Vascular xanthine oxidase and cytochrome P45 0 389 epoxygenases seem to be less important, since expression and activity 390

of these systems is not altered with ageing in humans [100]. 391

With biological ageing, mitochondria become dysfunctional leading 392

to reduced energy production and increased ROS formation. Mecha- 393

nisms related to mitochondrial dysfunction during ageing include 394

decreased ATP synthesis, increased apoptosis and mutations of mito- 395

chondrial DNA by oxidation [101]. During ageing, the electron flow in 396

mitochondria decreases, altering the oxygen consumption and inducing 397

ROS generation [101]. The pro-oxidative environment increases mito- 398 chondrial DNA damage, leading to further dysfunction of the respiratory 399

chain and more ROS production. Consequently, the rate ofapoptosis in- 400 creases, releasing an excessive amount of ROS into the cytosol, further 401

contributing to oxidative stress and vascular cell damage. 402

7.8. Endoplasmic reticulum stress in vascular ageing 403

Prolonged perturbation of the endoplasmic reticulum (ER) leads to 404 ER stress and unfolded protein response (UPR) and contributes to path- 405 ogenic processes associated with vascular damage and endothelial dys- 406

function [102]. The ER is an important site where proteins are folded 407 and post-translation modifications occur. It is also a site for Ca2+ storage 408

and cholesterol/lipid biosynthesis. Due to the large amount of unfolded 409

protein in the ER, a control system that avoids protein aggregation and 410

accumulation ofunfolded proteins is necessary. In experimental models 411 of ageing, the expression and activity of ER chaperones or folding 412

enzymes decay, whilst oxidative damage, such as carbonylation, is 413

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Vascular aging^Hypertension

Fig. 3. Role of reactive oxygen species (ROS) in vascular processes associated with ageing and hypertension. Pro-hypertensive factors, such as angiotensin II and endothelin-1, and biological ageing, increase ROS production in vascular cells. An increase in the levels of ROS lead to oxidation of proteins and DNA, affecting cell signalling and inducing injurious responses, such as inflammation, senescence, fibrosis, calcification, and hypertrophy in the vasculature. Oxidation of transcription factors that regulate the anti-oxidant capacity in vascular cells, such as Nrf2, are also affected by oxidation leading to decreased activity. Sources responsible for the increase in ROS generation and oxidative modification of cellular molecules are the mitochondria, NADPH oxidases (Nox) and endoplasmic reticulum (ER) stress.

exacerbated, leading to accumulation of misfolded/unfolded proteins and ER stress. This activates signalling mechanisms that are part of the UPR Induction of ER stress leads to endothelial cell apoptosis, but not senescence, implicated in endothelial dysfunction in ageing [103,104]. Inhibition of ER stress has been suggested as a novel therapeutic strategy to ameliorate vascular dysfunction during ageing [105]. However such approaches still require further investigation.

7.9. Vascular changes in hypertension recapitulate those in ageing

Many of the signalling pathways associated with vascular changes during ageing are also activated in hypertension leading to endothelial dysfunction, vascular inflammation, remodelling and increased arterial stiffness. With normal physiological ageing the process is gradual and regulated but in itself represents a strong and independent risk factor for hypertension and future cardiovascular events [106]. In susceptible individuals, due to genetic, environmental or in-utero factors 9fetal programming), processes underlying vascular changes are accelerated leading to 'early vascular ageing', which predisposes to cardiovascular disease. Numerous risk factors amplify the process of arterial ageing, including atherosclerosis, smoking, increased sodium intake and hypertension, due, in part, to increased oxidative stress, activation of proinflammatory and pro-fibrotic signalling pathways and upregulation of the renin-angiotensin-aldosterone system. As with ageing, experimental and human hypertension show a reduction in endothelium-dependent vasodilation, decreased NO bioavailability, NO synthase uncoupling, increased oxidative stress, telomere shortening and associated endothelial dysfunction. In arteries from aged humans, non-human

primates and rodents, expression of the AT[R is increased and sensitiv- 440

ity of the mineralocorticoid receptor to aldosterone is enhanced, 441

phenomena that are also observed in hypertension [107-109]. Ang II 442

promotes vascular calcification, inflammation, cell proliferation and 443

fibrosis and mimics age-associated vascular remodelling in young ro- 444

dents [110-112]. In large arteries these molecular and cellular processes 445

manifest as increased arterial stiffness, which is a major contributor to 446

elevated central blood pressure leading to isolated systolic hyperten- 447

sion, common in the elderly. Exactly what triggers these cellular and 448

vascular events remains unclear, and it is difficult to dissect out the 'age- 449

ing effect' from the 'blood pressure effect'. This 'conundrum of arterial 450

stiffness, elevated blood pressure and ageing' has recently been 451

reviewed by AlGhatrif and Lakatta [113], who concluded that vascular 452

properties depend on the net effect of multiple factors that are interde- 453

pendent and which change with ageing over a lifetime. 454

7.10. Effects of pro-hypertensive stimuli on vascular ageing: the renin- 455

angiotensin-aldosterone system (RAAS) 456

The RAAS plays an important role in functional, structural and 457

mechanical changes of the vasculature that occur with ageing and hy- 458

pertension [114]. This occurs through increased signalling via the AT 459

receptor. Expression of various components of the RAS, including 460

angiotensinogen, chymase, angiotensin converting enzyme (ACE) and 461

the AT1 receptor is increased in arteries of aged rodents and humans 462

[8,9]. To further support a role for the RAAS in the ageing process and 463

during hypertension are studies showing that ACE inhibitors and AT re- 464

ceptor blockers decrease ageing-associated vascular damage. Mice 465

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treated with enalapril or losartan demonstrated vasoprotection and an increase in life span [115,116]. Processes associated with these effects involve upregulation of NOS activity and increased NO production. An increase in antioxidant defences, such as SOD and glutathione, is another mechanism involved in the anti-ageing effects of the inhibition of the RAAS system, leading to an increase in NO bioavailability [117]. Moreover, lifelong treatment of young stroke-prone spontaneously hypertensive rats, with AT1 receptor blockers doubles the lifespan by improving endothelial function and alleviating complications of hypertension [118]. To further support a role for Ang II/AT1 receptor in oxida-tive stress, vascular injury and ageing, studies in mice with targeted disruption of the Agtr1a gene, which encodes the AT1A receptor, resulted in prolonged life span [119]. Agtr1a —/— mice developed less cardiac and vascular injury and oxidative damage was reduced compared with wild-type counterparts. The longevity phenotype was associated with an increased number of mitochondria and upregulation of pro-survival genes.

Clinical and experimental studies demonstrate that many pro-hypertensive systems influence processes of vascular ageing, including aldosterone, ET-1 and growth factors [120-124]. Arteries from aged rodents demonstrate upregulation of these systems, leading to stimulation of signalling pathways, oxidative stress, and activation of pro-inflammatory transcription factors, which promote a shift of endothelial and vascular smooth muscle cells to an ageing phenotype. On the other hand, infusion of Ang II, aldosterone or ET-1 in young animals, recapitulates arterial changes observed in aged animals.

7.11. Effects of novel anti-hypertensive factors on vascular ageing

NO is a potent vasodilator produced by endothelial cells that mediates vascular relaxation and thus plays a critical role in the regulation of blood pressure. Abnormalities in endothelial production of NO occur in hypertension and are due, in large part, to decreased eNOS activity [125]. NO donors such as glyceryl trinitrate (GTN) have been shown to possess anti-hypertensive properties [126] and evidence is emerging that NO and NO donors could confer beneficial effects on the phenotypic alterations that occur in the vasculature with ageing. For example, NO prevents differentiation of VSMCs into osteoblastic cells by inhibiting TGF-p [127]. The NO donor S-nitroso-penicillamine significantly reduces endothelial cell senescence and age-dependent inhibition of telomerase activity [128,129].

The gaseous messenger hydrogen sulphide (H2S) produced by cystathionine g-lyase (CSE) or cystathionine b-synthase (CBS) has recently emerged as a novel antihypertensive factor based on the observations that exogenous H2S is vasoprotective in pulmonary hypertension [130] and that it reduces systemic blood pressure by improving endothelial function [131]. CSE-deficient mice, have increased blood pressure and impaired endothelial function [132]. Mouse embryonic fi-broblasts from CSE knockout mice display accelerated cellular senescence and increased expression of p53 and p21, processes which were prevented by NaHS treatment. NaHS also enhanced Nrf2 nuclear translocation, and stimulated mRNA expression of Nrf2-targeted downstream anti-oxidant genes in this system, highlighting an important interplay between cellular ageing, senescence and oxidative stress [133]. Attenuation of endothelial cell senescence by H2S occurs through modulation of SIRT1 activity [134].

Plasma levels of H2S in humans decline with age [135] and several studies have shown that H2S protects against free radical-induced damage and exerts beneficial effects on age-associated diseases [136]. Several lines of evidence indicate that these beneficial effects may extend to vascular ageing, characterised by positive effects on many of the phenotypic vascular alterations that occur with advancing age. For example, the production of H2S is decreased in a rodent model of vascular calcification with the addition of H2S ameliorating this phenotype [137]. Further, in vascular smooth muscle cells, H2S was found to inhibit

calcium deposition in the extracellular matrix and suppress induction of osteoblastic transformation genes [138].

In endothelial cells stimulated with TNF-a, NaHS (H2S donor) suppressed pro-inflammatory responses by reducing the TNF-induced increase in expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin and E-selectin. Furthermore, TNF-a-induced NF-kB was decreased in the presence of NaHS [139]. H2S donors (NaHS and Na2S) can inhibit leukocyte adherence in mesenteric venules whilst inhibition of endogenous H2S synthesis promotes leukocyte adhesion and vascular inflammation [140].

7.12. Vascular damage in hypertension may be independent of ageing

Although there are many signalling pathways and functional and structural characteristics that are common in vessels during ageing and hypertension, these processes are dynamic and change throughout life and as such may not necessarily be superimposable. For example, with advancing age arterial stiffness and blood pressure start to diverge rather than parallel [141]. Also, circulating markers of inflammation including sVCAM-1, IL-6 and MCP-1 increase with age but do not necessarily correlate with elevations in blood pressure [142]. Furthermore, with ageing, aortic calcification is independently predictive of subsequent vascular morbidity and mortality beyond established risk factors with no evident correlation between calcification and systolic BP [143].

There is also some evidence to show that structural alterations in the vascular wall occur before the development of hypertension. For example rates of pulse wave velocity (PWV) increase are accelerated with both advancing age and elevated blood pressure. However, the effect of blood pressure on PWV increase occurs primarily during the prehypertensive phase and suggests that these vascular alterations precede the phase of established hypertension [8,144].

7.13. Lessons learned from children with hypertension

Vascular changes that occur with ageing may be independent of biological ageing in hypertension. This is highlighted in studies that have examined vascular function and arterial structure in children with hypertension. Endothelial dysfunction, arterial stiffening and structural alterations of the arterial wall may precede evidence of high blood pressure, as quantified by systolic and diastolic blood pressure, and may be independent of the ageing process [145]. This is evidenced by the findings that vascular injury is already present in children with mild hypertension, processes that are exaggerated as hypertension becomes more severe [146,147]. Functional alterations, including reduced endothelium-dependent vasorelaxation and decreased elasticity, seem to precede vascular structural changes. In obese pre-pubertal children, impaired brachial endothelial and vascular smooth muscle function is present without concomitant increase in carotid intima-to-media thickness. Functionally these changes lead to decreased vascular distensibil-ity and increased rigidity or stiffness. Arterial stiffness, as assessed by measurement of PWV, is increased in children with type 1 diabetes and in children with hypertension. Increased arterial stiffness in childhood hypertension is an important risk factor for severe hypertension and cardiovascular complications later in life. Results from the Amsterdam Growth and Health Longitudinal Study indicate that individuals with stiffer carotid arteries at 36 years of age were characterised during adolescence by increased blood pressure and increased PWV [148]. Factors that have been implicated in vascular dysfunction in childhood hypertension include activation of the sympathetic nervous system, adipokines, upregulation of the RAAS and increased oxidative stress, processes that also underlie physiological vascular ageing and EVA in adult hypertension [149-152].

Taken together, emerging experimental and clinical evidence indicates that although the molecular and cellular processes that characterise the vascular phenotype in hypertension resemble those that occur with normal healthy ageing, age per se may not be a critical

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591 factor. However, ageing may be a compounding factor that amplifies

592 vascular injury that occurs with blood pressure elevation. In the

593 presence of other co-morbidities, such as diabetes, dyslipidemia,

594 smoking and obesity, these processes may be further exaggerated.

595 8. Summary and conclusions

596 Ageing is associated with a progressive deterioration in endothelial

597 function, vascular remodelling, inflammation and increased arterial

598 stiffness. Processes underlying these processes include activation of

599 pro-inflammatory transcription factors, oxidative stress, cell senescence

600 and apoptosis, aberrant signalling cascades and a shift from a vasocon-

601 strictor to a proliferative vascular cell phenotype. Many of these

602 phenomena are also relevant in the pathophysiology of hypertension,

603 which is characterised by a vascular phenotype of impaired

604 endothelium-dependent vasorelaxation, arterial remodelling, increased

605 stiffness and vascular inflammation. Through such vascular changes,

606 ageing and hypertension are closely interlinked: ageing promotes

607 hypertension and pro-hypertensive factors promote vascular ageing.

608 Whilst many of the molecular processes and signalling pathways

609 contributing to vascular dysfunction are common in ageing and in hy-

610 pertension, biological age per se, may not be a fundamental factor,

611 since vascular damage is already present in children and young adults

612 with hypertension. A better understanding of the vascular biology of

613 ageing will facilitate development of strategies to promote healthy

614 vessels and suppress age-associated changes, especially in pathological

615 conditions. Such approaches could prevent or ameliorate vascular

616 damage in hypertension and hence reduce cardiovascular diseases,

617 commonly linked to ageing.

618 Disclosures

619 There are no disclosures to declare. Q4 Acknowledgements

621 Work from the author's laboratory was supported by grants 44018

622 and 57886, from the Canadian Institutes of Health Research (CIHR)

623 and grants from the British Heart Foundation (BHF). RMT is supported

624 through a BHF Chair. ACM is supported through a Leadership Fellowship

625 from the University of Glasgow.

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